The rotavirus VP7 neutralization antigens of serotypes 1,2,3 and 4 were analyzed for genetic relatedness by hybridization of their genes using single human rotavirus gene substitution reassortants. Double-stranded (ds) genomic RNAs extracted from tissue culture adapted human rotaviruses (serotypes 1-4) were hybridized to single-stranded (ss) mRNA probes derived from human-bovine rotavirus reassortants representing serotype 1,2,3 or 4 which contained only the VP7 gene of their human rotavirus parent. When stringent conditions were used, bovine rotavirus genes did not hybridize to the corresponding genes of human rotaviruses and thus hybridization occurred only between the human rotavirus VP7 gene in the labeled probe and a related VP7 gene present in the human rotavirus under study. A high degree of homology was demonstrated between the probe and the VP7 gene of strains belonging to the same serotype, whereas hybrid bands did not form with VP7 genes from human rotaviruses belonging to a different serotype. This technique was also used to examine RNA extracted from stools of 15 children hospitalized with rotavirus diarrhea. All five viruses with a "short" RNA pattern shared homology with the DS-1 strain (serotype 2) VP7 gene; two of them had been previously adapted to tissue culture and shown to be serotype 2 strains by neutralization. Of the remaining 10 viruses with "long" RNA patterns, two hybridized to the D strain (serotype 1) VP7 gene, six hybridized to the P strain (serotype 3) VP7 gene, and two hybridized to the ST3 strain (serotype 4) VP7 gene. The stool specimens from 14 of these 15 children were also tested by ELISA with VP7 serotype-specific monoclonal antibodies directed at human serotype 1,2,3 or 4. Ten specimens were successfully serotyped with this method and there was complete concordance with the hybridization assay. It appears that serotype identification by hybridization with the reassortant probes is both sensitive and specific and circumvents the need for tissue culture adaptation of human rotaviruses since the genomic RNAs are purified directly from stool specimens.